FI83972B - α-acetolactate decarboxylase enzyme and preparation thereof - Google Patents

Description

! 83972 α-Acetolactate decarboxylase enzyme and its preparation

Divided separated from application 842203.

This invention relates to a novel α-acetolactate decarboxylase and to a process for its preparation.

During the fermentation of alcoholic beverages, e.g. beer or wine, small amounts of diacetyl are often formed. The formation of diacetyl is very disadvantageous because it has a strongly unpleasant odor and, in the case of beer, even amounts of diacetyl as low as about 0.10-0.15 mg / liter adversely affect the smell and taste of the beer.

The presence of diacetyl in beer is due in part to the Pediococ-cus strain, which forms directly diacetyl (E. Geiger, Diacetyl in Bier, Brauwelt 46, 1680-1692, 1980) and in part to the fact that brewer's yeast from pyruvate forms α-acetolactate, which changes in a non-enzymatic but temperature dependent reaction to diacetyl. During the maturation of beer, diacetyl is converted to acetone by reductases in yeast cells. Acetoin can be accepted in beer at much higher concentrations than diacetyl in terms of taste and odor.

25

Another possibility to reduce the amount of diacetyl in beer is to directly convert α-acetolactate to acetoin by means of acetolactate tidecarboxylase, cf. EP patent application No. 46066. Acetolactate decarboxylase can be added during the main fermentation of the beer or during its maturation phase.

This enzyme is an intracellular enzyme and is obtained from the microorganism Klebsiella pneumonia (E. Juni, J. Biol.

35 Chem. 195, 715-726, 1952). However, since Klebsiella pneumonia is a pathogenic microorganism, it is not very suitable for use in industry. In addition, the acetolactate decarboxylase produced by this microorganism is poorly preserved under its intended conditions of use, i.e., in the case of beer, at a pH of about 4.3 and about 10 ° C.

It is an object of the present invention to provide a novel aceto-5-lactate decarboxylase enzyme which has better stability under the above-mentioned conditions and which can also be obtained from non-pathogenic microorganisms and with better yields.

The invention is based on the surprising finding that a novel acetolactate decarboxylase having such properties is produced in very large quantities by a microorganism belonging to the species Bacillus licheniformis.

The above-mentioned EP patent application states that enzymes that cleave acetolactate are also obtained from enzymes other than Klebsiella pneumonia. However, this has not been demonstrated and it does not appear from the publication that the α-20 acetolactate decarboxylase with good properties of the above invention could be prepared in high yields from Bacillus licheniformis.

According to a first aspect of the present invention, there is provided a novel stable α-acetolactate decarboxylase enzyme, characterized in that it has a pH optimum in the range of 5-6 and is identical to that of an α-acetolactate decarboxylase enzyme obtained by culturing in a suitable carbon. and Bacillus licheniformis strains ATCC 30 12759, ATCC 12713, ATCC 11946, ATCC 27326, NRRL B-3751, NCTC 2120, NCTC 8721, NCIB 6816, NCIB 8537 or NCIB 11868 in a medium containing nitrogen sources and inorganic salts.

The α-acetolactate decarboxylase enzyme product may be in solid or liquid form and usually has an effect of the order of 0.1-10 NU (to be defined) per mg of protein in solid form.

I! 3,83972

According to another aspect of the present invention, an α-acetolactate decarboxylase enzyme can be prepared according to a method comprising culturing in a suitable medium containing carbon and type sources and inorganic salts an α-acetolactate decarboxylase-producing Bacillus licheniformis acetol strain followed by a strain of cells in the medium.

Preferred strains of Bacillus licheniformis are ATCC 12759, ATCC 12713, ATCC 11946, ATCC 27326, NRRL B-3751, NCTC 2120, NCTC 8721, NCIB 6816, NCIB 8537 and NCIB 11868 and their α-acetolactate decarboxylase enzyme producing mutants and variants.

15

When a Bacillus licheniformis strain is used to prepare the α-acetolactate decarboxylase enzyme of this invention, the medium should preferably contain a nitrogen source whose free amino acid content does not inhibit the formation of α-acetolactate decarboxylase, preferably inorganic.

The microorganisms required to prepare the α-acetolactate decarboxylase enzyme of this invention have been selected for their ability to convert α-acetolactate to acetoin, which is then detected.

More than 300 Bacillus strains have been tested according to the following method: The strains are cultured in shake flasks at 30 ° C and 37 ° C for 24 h in the following BCM substrate: K 2 HPO 4 1 g

NaH 2 PO 4, 2H 2 O 1 g 35 (NH 4) 2 SO 4 2.5 g

NaCl 0.25 g

MgSO 4, 7H 2 O 0.2 g

FeCl 3, 6H 2 O 0.04 g 4,83972

MnSO 4, H 2 O 0.25 mg

Glucose 10 g

Yeast extract 3 g

Water ad 1,000 g 5

After culturing, the cells were harvested by centrifugation, washed with 0.03 M phosphate / citrate buffer (pH 6.0) and resuspended in the same buffer until OD (450) 40. Aliquots of 2 ml were sonicated or treated with 1 mg. with lysozyme per ml at 37 ° C for 30 min and then centrifuged.

The α-acetolactate decarboxylase activity of the supernatants was determined according to the method described in section 15 "Determination of the α-acetolactate decarboxylase activity".

The screening method revealed that only a few of the approximately 300 microorganisms tested produce detectable amounts of α-aceto-20 lactate decarboxylase. These microorganisms are listed in Table 1 below, as well as their enzymatic activity as determined by the above method.

25 Table 1

Result of the screening method

Strain Strain (NOVO-Effect (NU) Protein collection no.) Per mg protein mg / ml 30 B. brevis A303 0.13 4 B. coagulans A 345 0.007 3 B. pumilus A 185 0.008 2 B. pumilus A 328 0.011 3 35 B. pumilus A 329 0.004 2 B. subtilis A 518 0.005 3 B. subtilis C 601 0.06 1 B. licheniformis A 446 0.11 3 5 83972

Table 1 (continued)

Result of the screening method

Strain Strain (NOVO-Effect (NU) Protein 5 collection no.) Per mg protein mg / ml B. licheniformis C 600 0.19 2 B. licheniformis A 88 0.15 2 B. licheniformis A 105 0.13 2 10 B. licheniformis A 106 0.15 2 B. licheniformis A 200 0.15 2 B. licheniformis A 203 0.25 2 B. licheniformis A 244 0.11 2 B. licheniformis A 248 0.14 2 15 B. licheniformis A 1240 0.13 2

Strains C 600 and C 601 were deposited with the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, on 27 May 1983 and have been assigned ha-20 numbers NCIB 11868 and NCIB 11869, respectively.

Other strains have been obtained from different culture collections, as can be seen from the following list: 25 NOVO no ._Search number A 303 ATCC 11031 A 345 NRS 83 A 185 ATCC 71 A 328 ATCC 4520 (B. Mesentericus var. Flavivus) A 329 ATCC 7065 A 518 IAM 1109 (B. natto) A 446 NRRL B-3751 (Defined by the applicants as B. licheniformis) 35 A 88 ATCC 12759 A 105 ATCC 12713 = NRRL B-1001 A 106 ATCC 11946 A 200 NCIB 6816 6 83972 NOVO -n: o_Search number A 203 NCIB 8537 A 244 NCTC 2120 A 248 NCTC 8721 5 A 1240 ATCC 27326

The prior art shows (E. Juni, same as above) that cell-free extracts of the microorganism Bacillus subtilis are capable of decarboxylating? -Acetolactate to form acetone, but without the use of these extracts. Therefore, 69 different strains of Bacillus subtilis have been tested. Of these, only 5 showed α-acetolactate decarboxylase activity and, in addition, these five strains were all poor enzyme manufacturers. Strains of Bacillus coagulans and Bacillus pumilus 15 also proved to be too poor producers of the enzyme in industrial use.

Of the Bacillus licheniformis strains tested, 13 did not produce detectable amounts of α-acetolactate decarboxylase, 21 produced 20 enzymes in very low amounts, and 11 strains (10 of which are listed in this specification) produced large amounts of the enzyme. It was found by sequential cross-linked immunoelectrophoresis that all the listed Bacillus licheniformis strains produce the α-acetolactate decarboxy-25ase enzyme, which is immunochemically identical to that produced by Bacillus licheniformis strain C 600.

Bacillus brevis strains have been grown in three different media in shake flasks. Only one strain, A 303 (ATCC 11031) 30, showed α-acetolactate decarboxylase activity in all three media. The other 11 strains did not show significant α-acetolactate decarboxylase activity in any of the three media used. The α-acetolactate decarboxylase enzyme-35 produced by Bacillus brevis A 303 was found to be non-identical in terms of immunochemical properties of the enzyme produced by Bacillus licheniformis strain C 600. However, the fact that α-acetolactate decarboxyl 7 83972 cylase enzymes produced by different Bacillus licheniformis strains have immunochemical identity suggests that other α-acetolactate decarboxylase-producing Bacillus brevis A strains 303. As regards "immunochemical identity", reference is made to NH Axelsen et al. "A Manual of Quantitative Immunoelectrophoresis" (Oslo 1973), paragraph 10.

In the practice of this invention, Bacillus brevis differs from Bacillus strain licheniformis in that α-acetolactate decarboxylase can be prepared from Bacillus brevis without undesired side effects, specifically the ferric acid decarboxylase effect produced by all tested Bacillus licheniformis. Ferrulic acid decarboxylase decarboxylates ferric acid, which is present in fermented beer, to a very bad-tasting compound, 4-vinyl guaiacol. Therefore, ferric acid decarboxylase must be removed from the culture medium of Bacillus licheniformis during the recovery process.

Bacillus brevis differs from Bacillus licheniformis further in that Bacillus brevis produces an enzyme which converts the normal precursors of 2,3-pentanedione in beer to 3-hydroxy-2-pentanone and thus reduces the levels of malodorous pentanediones to a suitably low level. Bacillus lichenifor-25 mis strains do not produce this enzyme.

Determination of alpha-acetolactate decarboxylase activity One unit of NOVO (NU) is defined as the amount of enzyme which produces 1 μπιοί acetone per minute at 20 ° C and pH 30 6,0 (0,03 molar citrate / phosphate buffer) when incubated. with an α-acetolactate substrate in the following test:

Substrate: 35 α-acetolactate substrate was prepared immediately before use by hydrolysis of alpha-acetoxy-alpha-methyl-aceto-acetic acid ethyl ester: 8 83972 30 μΐ diester (0.165 mmol), 240 μΐ water and 330 μΐ 1 M NaOH were mixed and stored on ice 15 min. 5.4 ml of water were then added and the resulting hydrolyzate ti was used for the test.

5

Test;

The buffer and test samples with an enzyme activity of about 0.006-0.3 NU were mixed together and heat treated at 20 ° C for a few minutes (final volume 10.0 10 ml). At time t = 0, 400 μΐ hydrolyzate was added and 1 ml samples were taken at time t = 3, 6, 9, 20 and 50 min. The enzyme reaction was stopped by placing the samples on ice and adding 200 μΐ of 1 M NaOH.

The amount of acetone formed is measured according to W. W. Westerfeld (A Colorimetric Determination of Blood Acetoin, J.

Biol. Chem. 161, 495-502, 1945) by adding 0.2 ml of 0.5% creatine and 0.2 ml of 5% alpha-naphthol in 2.5 N NaOH (freshly prepared) to 1.2 ml of the above 20 samples available. After a reaction time of 60 min, E (524) was measured.

For comparison, a sample with buffer instead of cell extract was used.

25 Standard curves were plotted for 0, 0.5, 2 and 4 μΐ / ml acetone.

Preparation of alpha-acetolactate decarboxylase enzyme product

The Bacillus strain capable of producing the α-acetolactate decarboxylase enzyme of the present invention is usually allowed to grow in a suitable solid nutrient solution at about 30 ° C before being cultured under aerobic conditions in a suitable fermentation medium. The fermentation medium contains assimilable carbon sources (e.g. dextrose) and a base salt mixture with e.g. ammonium sulfate as a nitrogen source. In order to obtain high yields in the Bacillus licheniformis system, it is important that the fermentation medium does not contain excess free amino acids that can suppress α-1! 9,83972 acetolactate decarboxylase production. The fermentation is carried out at about 30 ° C and a pH of 7, which is maintained almost constant by automatic means. Ventilation and agitation are adjusted to provide a positive oxygen pressure.

After fermentation, the cells are harvested by centrifugation and washed with a suitable buffer. The cells are lysed by sonication, French pressure treatment, lysozyme treatment, Manton-Gaulin method, or a combination of these methods.

After centrifugation, a crude extract is obtained which may be subjected to a purification process, as described in the next section.

15 Purification of alpha-acetolactate decarboxylase enzyme

The α-acetolactate decarboxylase enzyme of the present invention can be purified from the crude extract by combining one or more of the following steps: a) ammonium sulfate precipitation b) polyethylene glycol precipitation c) DE 52 anion exchange chromatography and dialysis and dialysis and diacidation lyophilization g) phenyl sepharose chromatography h) chromatofocusing Purification of α-acetolactate decarboxylase obtained by culturing Bacillus licheniformis strain C 600 as described in Example 1 was performed as follows: 94 g of cells (wet weight) were suspended in 20 mM K-phosphate 8 and lysozyme treatment was performed for 30 min 35 using 1 mg / ml lysozyme at 37 ° C and then sonicated on a Branson B 12. After centrifugation, the supernatant was applied to a DE 52 anion exchange column, whereupon the enzyme was completely adsorbed. The column was washed with 10,83972 20 mM potassium phosphate buffer (pH 6.8) and eluted with a potassium phosphate gradient (20 mM to 500 mM), pH 6.8. Fractions were analyzed for enzyme activity as described above. The effect fractions were pooled.

5

The combined fractions were acid precipitated by the addition of phosphoric acid to pH 4.5. After centrifugation, the supernatant was dialyzed against 20 mM potassium phosphate pH 6.8. The dialysate was applied to a hydroxyapatite (HA) column, which was washed with potassium phosphate buffer pH 6.8 and then eluted with a potassium phosphate gradient as above.

Fractions showing enzyme activity were pooled and dialyzed overnight with 20 mM potassium phosphate, pH 6.8.

Finally, the dialysate was lyophilized.

The enzyme activity and yields are shown in the following table: H 83972 tn

O

G -P

0) 0 o oo * r ro «- vo

C 3 O (N · * Τ CM OS OS O

• HE-IC ^ T— r— s— r- s—

E

<a -P i tn tn H 3

Ό -PC

X tn -p

3 -PO

α ό p p Tr mm m oo · * τ »* 30) * ** ^ *» v

-. CU PC r- Ό · s— CN CM CO CM

0 3 (N <n rs) es) ro

C H

• H C

G -H

Cu σι-h OEOm o r ^ rsj cm »to 4J u n t— corn vo as <- • P P O o t— mm mmoo CU D 1) P ^ ~ - C Z C Qj o O OO ooo

C

σ '.3 • <3 · .3 σ> -p

Pi - Pi C -pcm vo ms— voom CU D 3 o oo ro r ~ r-'OT— • ro 2 Λ! ro co -sr ro cm cm ro 3

M «H

M O

tn o i — i λ; C S oo S- OVO vorr p; CU coco mro cmoo 3 -P P oo -sr -sr cm cm <- • P L> L3 3 * 302: 0.0 s- s- s- T- .-

3 P

E · o Ή c c

-P -P

tn -p -PQ) oo o oo s— co o .3 -P VO <- s— vo as oo co PO s— m Covo sr sr ro (UPO cm co e. c. ε s—: 3

-P

tn -p> s E tn • P> 1 <U>. co • ei.-π o o oo mmo - I— (OP VO C- o O CM ST ·.

-P> £ co CM COCO CM CM T—> c Cl

<D PJ

O Q) 3 C I

O -P Ai -P -PO -PO

md (U ^ rP ^ tnoC> i -P C

4J 4J + j: 3 iUJ OM P -P -P 3 P

U O 3 (D O-π Q) 03rP ti) C CU 0 E C C tn C

P 3 -P -P + J -p tn: 3> i CL) -P -PO 3 <U> .0) 3 -P 3 tnu-) CM inu on> itu tn-pppo mi o -p pc p: pp; m ppcq. p pc p m p; p p; h p! x C 3 3 Ό 3 Ό 3 0. C 3-P Ό 3 I 3 * P 3 P 3 3 P 3 x p η X p 3 CU -P '-3 C P <P W -P: 3 3 X C CtC>. Ui Q>. P C tn Q-co LhP X σ · η Q-ro i-j i2 83972

Chemical properties of the enzyme

The pH dependence of the effect of the Bacillus licheniformis acetolactate decarboxylase enzyme of the present invention was determined from extracts of Bacillus licheniformis strains 5 A 446 and C 600 at different pH values in 0.03 M citrate / phosphate buffer at 20 ° C from the above a-acetolac according to the method of determination. Reference is made to the accompanying drawings, in which Figure 1 and Figure 2 graphically show the relative effect on pH-10 with α-acetolactate decarboxylase enzymes prepared from strains A 446 and C 600, respectively. The optimum pH was found to be between 5-6.

Stability The stability of the crude extracts of Bacillus licheniformis was tested under the conditions of use, i. At 10 ° C and in fermented but not matured beer.

The stability is shown in the following table.

20

Table III Stability test

Strain Half-life in days 25 A 446 21 C 600 15 A 446 11.5 (determined from fermented beer) 30 When the half-life of Klebsiella pneumonia is only 2-3 hours, the enzyme of the present invention shows significantly better stability under beer loading conditions. to a known enzyme.

The 35 μl assay is determined to be about 4.7 from the crude extract of strain A 446 by thin-layer gel electrofocusing.

i3 83972

Immunological Properties The purified fraction of α-acetolactate decarboxylase from strain C 600 (Bacillus licheniformis) was used to immunize rabbits according to the method described by Harboe and Ingild: 5 N. H. Axelsen: Handbook of Immunoprecipitation-in-Gel Techniques, Blackwell Scientific Publications, London 1983.

The antibody thus prepared for Bacillus licheniformis was used to perform cross-immunoelectrophoresis and sequential cross-immunoelectrophoresis, as described by A. 0. Grubb and J. Krtf11, respectively, in the above-mentioned publication.

When the antigen is not homogeneous, the antibody produced is 15 polyspecific and generates up to 15 bands in cross-immunoelectrophoresis, one of which is the α-acetolactate decarboxylase precipitation band. To identify this band, an upper layer technique based on the effect of the enzyme was followed. after immunoelectrophoresis, the plate was not fixed and stained as usual, but was incubated for 5 min in 30 ml of the following mixture in a 14 cms diameter petri dish lid: 150 μΐ ethyl-2-acetoxy-2- Mixed and incubated with methyl acetoacetate at room temperature for 15 min .

25 1 200 μΐ H 2 O H 2 O was then added to 1 650 μΐ 1 N NaOH to 30 mL.

After incubation, the mixture was allowed to drip off a plate wrapped in Vitawrap plastic film and aluminum foil and incubated for 30 min at room temperature.

The plate was then placed on the lid of a 14 cm diameter petri dish and covered with the following mixture: 35 30 ml of 2% LSA agarose (HaO) 3 ml of 1% creatine (Ha0) Stirred at 55 ° C i4 83972 6 ml of 5% naphthol In 2.5 N NaOH

The covered plate was incubated at room temperature for about 1 h. The α-aceto-5 lactate decarboxylase precipitate band is red in color and can be identified.

As mentioned above, sequential cross-immuno-10 electrophoresis of all listed Bacillus licheniformis enzymes with the C 600-α-acetolactate decarboxylase antibody showed that the Bacillus licheniformis enzymes from Bacillus licheniformis are not immunochemically identical to those from α-acetolac precipitates with an antibody to C 600-α-acetolactate decarboxylase, indicating that strains of Bacillus licheniformis and Bacillus brevis both produce different types of α-acetolactate decarboxylase enzyme, but are well suited for their intended use in beer production.

The following examples are provided as illustrative embodiments of the present invention and are not intended to be particularly limited thereto.

Example 1 Strain A 446 (Bacillus licheniformis, NRRL B-3751) was grown in TY medium in shake flasks at 30 ° C until the OD (450) was 6.

TY medium: 30 Trypticase 20 g

Yeast extract 5 g

FeCl 3, 6 HaO 7 mg

MnCl 2, 4 HaO 1 mg

MgSO 4, 7 HaO 15 mg 35 Distilled water ad 1000 ml is 83972 100 ml of shake flask culture was transferred to a 1.5 liter

Kieler fermentation vessel containing medium containing inorganic salts per liter: 5 CaCl2, 2 H2O 0.5 g

MgSO 4, 7 H 2 O 0.5 g KH 2 PO 4 4.0 g (NH 4) 2 SO 4 22 g

Trace metal solution * 3 ml 10 Pluron 1 ml

Dextrose 150 g ♦ Trace metal solution: H3BO3 500 mg / 100 ml 15 CuSO 4, 5 H 2 O 63 mg / 100 ml KI 100 mg / 100 ml

FeCl 3, 6 H 2 O 333 mg / 100 mL

MnSO 4, H 2 O 448 mg / 100 mL

NaMoO 4, 2 H 2 O 200 mg / 100 ml 20 ZnSO 4, 7 H 2 O 712 mg / 100 ml

During the culture, the pH was maintained at 7.0 by the addition of NaOH. The temperature was 30 ° C and the ventilation and agitation were adjusted to obtain a positive oxygen pressure.

25

Cells were harvested by centrifugation after the cell density corresponded to OD (450) 34.

The progress of the fermentation is shown in the following table:

Table VI

Effect measured during fermentation Fermentation time NU / ml culture OP M50 ^ 35 hours 0 0 1.35 5 0 1.69 21 0.022 19 ie 83972

Table VI (cont

Effect measured during fermentation Fermentation time NU / ml culture OP (450) in 5 hours 24 0.076 28 28 0.440 34

Example 2 Bacillus licheniformis strain A 446 was propagated and cultured as described in Example 1, except that the fermentation medium contained 180 g of dextrose and that the cells were harvested when the cell density corresponded to OD (450) 47.

112 g of cells (wet weight) were suspended in 20 mM K-phosphate buffer, pH 6.8 and treated with lysozyme for 30 min using 1 mg / ml lysozyme at 37 ° C. The dissolved cells were then sonicated on a Branson apparatus B 12. 20 Ammonium sulfate was added to the resulting crude extract until 25% saturation and the mixture was placed on ice for 30 min.

After centrifugation, there was an enzyme effect in the supernatant.

25

Ammonium sulfate was added to the supernatant until 70% saturation and the mixture was placed on ice for 30 min. After centrifugation, there was enzyme activity in the precipitate.

30

The precipitate was suspended in 20 mM K-phosphate, pH 6.8, and then 200 mg / ml polyethylene glycol (MW 6,000) was added. The mixture was left at room temperature for 30 min. The effect was observed in the supernatant after centrifugation.

The protein content of the supernatant was adsorbed onto a Whatman DE 52 anion exchanger, which was then washed with 20 mM K-phosphate buffer, pH 6.8, and then eluted with a K-phosphate-35 83972 buffer gradient (20 mM to 500 mM) pH 6.8. Fractions with enzyme activity were pooled and dialyzed against 20 mM K-phosphate buffer, pH 6.8. Finally, the dialysate was lyophilized.

5

The progress of the purification is shown in the following Table VII: ie 83972 c 0) c

• rA

: 3 tn c O c -P 1) O: 3 o oj o in o m o- O c o i— o <—1 o o o- E <t »- r- 04 m m t— τ- Ι

UI

3

-P C

C / 5 t- * H 0 Ό 5-i lti o- Comoro -C 5-4 - ^ ^ ^ 30) -— oi m o- o - σ Λ λ; No)

• rA

c

R · I

CP-H

EOJ'i »ο σ rr ld ro rr

• P -— -r m 04 IOOOT

P Ο Ο Ο Ο T- r-

00) 5-4 - «. ·. - v ^ V

2 Oi ft o O O O Ooo c 05 05

•B

Λί

• H 00 * 3- -T vo CP -a · VO

O 04 oo oo, - 04 m o- 04 T VO O 'CM 04, -

M rH

-Hg vo m O 'i— and ^ r> σ m oo o in m 5-1 · = τ ο -τ m σ σ 0 D 0) - - - .. _

P2 ϋι O, - t—, - OO

3

3 -H

id c

E- <-H

*B

0) vo o oo moitnoo -p m σ n m σν 0 IA t— VO O '00 04 04 5-iCp · * τ (N i— m

Cu E - - Γ Η

E

> 1 CP

5> -i Ή Ο Ο Ο Ο Ο Ο τ- Ο 1 I vo vo vo in rr in *> E -u · 'ϊ · - <r in 040404

I III

II El C .22 I ft) Λί

H U) C I «3« 1 C I Q) .3 04- * 3 C

II: 3 103 W -H O 3> p U) 5-4) -4 4-1 5-4 -H

C 3 3 E E 3 3 3 C 3 3 3 -PH Λ5 Vi Λ -h U-iU) ο ή ί> E 3 ιλ λ; mno tn m> an 3 wc> - H 3 -H -h II — I WEIH 3> 1 O -P -P 04: 3 3 -P>, 3 Jp r-twc-H: 3 CE -η: 3 ρηλ oi>, m ^ "j>, h -P-P3 OCO-Pri 3 3 3 -P -ro O 0> ί O -P -P3

0 3C-P 3E-P 3-P -P Oi-H3C 3 W · H 3 -PC

• H 3 3 U): 3 E 3 C iH-HC3C: 3 CP U) 3 -PQP: 3 -ΡΌ3 P · 3 U) -H rH 3 3 3 3 I — I 0 3 3 3 rP 3 -H 3 u) Cn-ro U) 3 3

X M E H 4J 1 * PU) 3 Ό U) U-ι U) H44 C03i Η-Ρ0 -Η-ΡΛί JZ

3 3 C --4 3 in c η Λί d C · 4 hm: 3 tn 3 O -H Ό 0 -p C 3 O h 3 P 3 3 0: 3 3 3 3 3 3 3 0 3 3: 3 3 3 O: 3 -C -H 3> t -C Ή: 3 3 [p CXCCP O-CWW-P DCL-PWP t <CH «n> - + J c H> i -M -r — i 03

Claims (6)

Process for the preparation of the α-acetolactate decarboxylase enzyme according to claim 1, characterized in that the α-acetolactate decarboxylase-producing Bacillus Lich-15 eniformid α-acetolactol strain is prepared in a suitable medium containing carbon and nitrogen sources and inorganic salts, followed by cells in the medium. Method according to claim 2, characterized in that the Bacillus licheniformis strain is selected from the group consisting of ATCC 12759, ATCC 12713, ATCC 11946, ATCC 27326, NRRL B-3751, NCTC 2120, NCTC 8721, NCIB 6816, NCIB 8537, NCIB 11868 or mutants or variants thereof having substantially the same properties. 25 Process according to Claim 2 or 3, characterized in that the medium comprises a nitrogen source whose free amino acid content does not suppress the formation of α-acetolactate decarboxylase. 30 Process according to Claim 4, characterized in that the nitrogen source is an inorganic nitrogen source. 20 83972 1. α-acetolactate decarboxylase enzyme, which is shown to have a pH optimum of 5-6 and a genomic genome or a strain of Bacillus licheniformis ATCC 12759, 5 ATCC 12713, ATCC 11946, ATCC 27326, NRRL B-3751, NCTC 2120, NCTC 8721, NCIB 6816, NCIB 8537 or NCIB 11868 and that the heating medium is free of co- and quaternary chlorine and organic salt. 2. A method for the preparation of α-acetolactate decarboxylase enzyme according to claim 1, which provides for the production of α-acetolactate decarboxylase as a producer of Bacillus licheniformis or a warming medium of the same medium as the quaternary salt. tillvaratages ur cellerna i mediet. 3. A patent according to claim 2, which comprises a Bacillus licheniformis strain in groups 20 of ATCC 12759, ATCC 12713, ATCC 11946, ATCC 27326, NRRL B-3751, NCTC 2120, NCTC 8721, NCIB 6816, NCIB 8537, NCIB 11868 or a mutant or a variant of a mutant medium. 25 4. A preferred form of patent invention 2 or 3, which is used as an intermediate in a quaternary form, in particular from an amino acid underride in the image of an acetolactate decarboxylase. 30 5. For the purposes of claim 4, the provisions of this invention are incorporated into organic law.